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J. Biol. Chem., Vol. 279, Issue 24, 24944-24956, June 11, 2004

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A Novel "Clip-and-link" Activity of Repeat in Toxin (RTX) Proteins from Gram-negative Pathogens

COVALENT PROTEIN CROSS-LINKING BY AN ASP-LYS ISOPEPTIDE BOND UPON CALCIUM-DEPENDENT PROCESSING AT AN ASP-PRO BOND^*

Radim Osika, Kateina Procházková, Miroslav ulc, Irena Linhartová, Vladimír Havlíek, and Peter ebo

From the Institute of Microbiology of the Academy of Sciences of the Czech Republic, Vídeòská 1083, CZ-142 20 Prague 4, Czech Republic

Clinical isolates of Neisseria meningitidis produce a repeat in toxin (RTX) protein, FrpC, of unknown biological activity. Here we show that physiological concentrations of calcium ions induce a novel type of autocatalytic cleavage of the peptide bond between residues Asp⁴¹⁴ and Pro⁴¹⁵ of FrpC that is insensitive to inhibitors of serine, cysteine, aspartate, and metalloproteases. Moreover, as a result of processing, the newly generated amino-terminal fragment of FrpC can be covalently linked to another protein molecule by a novel type of Asp-Lys isopeptide bond that forms between the carboxyl group of its carboxyl-terminal Asp⁴¹⁴ residue and the -amino group of an internal lysine of another FrpC molecule. Point substitutions of negatively charged residues possibly involved in calcium binding (D499K, D510A, D521K, and E532A) dramatically reduced the self-processing activity of FrpC. The segment necessary and sufficient for FrpC processing was localized by deletion mutagenesis within residues 400–657, and sequences homologous to this segment were identified in several other RTX proteins. The same type of calcium-dependent processing and cross-linking activity was observed also for the purified ApxIVA protein of Actinobacillus pleuropneumoniae. These results define a protein cleavage and cross-linking module of a new class of RTX proteins of Gram-negative pathogens of man, animals, and plants. In the calcium-rich environments colonized by these bacteria this novel activity is likely to be of biological importance.

Received for publication, December 22, 2003 , and in revised form, March 15, 2004.

^* This work was supported by Grant Agency of the Czech Republic Grant 310/02/1448 and by Howard Hughes Medical Institute International Research Scholarship Award 55000334 (to P. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Inst. of Microbiology CAS, Vídeòská 1083, CZ-142 20 Prague 4, Czech Republic. Tel.: 420-241-062-762; Fax: 420-241-062-152; E-mail: sebo{at}biomed.cas.cz.

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